Cell biology and molecular genetics

Background: Hearing loss (HL) is the most common congenital defect in humans. One or two in thousand newborn babies have prelingual hearing loss. Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common form of hereditary deafness. Hearing loss is more common in the developing countries which is due to genetic and environmental (cultural -health factors) reasons. HL has a wide range of clinical demonstrations including: congenital or late onset, conductive or sensoryneural, syndromic or non-syndromic hearing loss. The goal of this project is to determine the portion of the DFNB21 (TECTA) in ARNSHL in families with negative GJB2 gene in Khuzestan province. Materials and Methods: We studied 21 families with ARNSHL with at least 4 patients and negative for GJB2 mutations from Khuzestan province. Genetic linkage analysis was performed using STR markers linked to DFNB21 locus. Results: Following genetic linkage analysis and haplotyping, out of 21 families with ARNSHL, o...

Abstract Hearing loss is one of the most common sensorineural disorders that occur in 1:1000. Mutation in the GJB2 (CX26) gene at the DFNB1 locus on chromosome 13q12 is the most important cause of congenital hearing loss. The aim of this study was to determine the hearing loss causative mutations in the GJB2 gene in a 37 year-old woman with non-syndromic congenital hearing loss. A compound heterozygous mutation (35delG/del120E) was found in the GJB2 gene. With regards to the variety of mutations in the GJB2 gene, screening the causative mutations of hearing loss is recommended for subjects referred to genetics counseling centers before marriage and pregnancy.

Background: Gastric cancer is one of the most common diseases of digestive system with a low 5-year survival rate and metastasis is the main cause of death. Multi-factors, such as changes in molecular pathways and deregulation of cells are involved in the disease development. Epidermal growth factor receptor pathway (EGFR) which is associated with cell proliferation and survival can influence cancer development. EGFR function is governed by its genetic polymorphism; thus, we aimed to study the tyrosine kinase domain gene mutations of the receptor in patients with gastric cancer. Methods: In this experimental study, 123 subjects (83 patients with gastric cancer and 40 normal subjects) were investigated in north of Iran for EGFR gene polymorphisms during 1 year. Genomic DNA was extracted by DNA extraction kit according to the manufacture’s protocol. Polymerase chain reaction single-stranded conformation polymorphism (PCR-SSCP) and silver staining were performed for investigating EGFR ...

TIM (T-cell immunoglobulin (Ig) and mucin domain)-1, one of the members of TIM family, expresses on Th2 cells and promotes the production of Th2 signature cytokines. This can increase a series of responses in these cells which could be one of the causes of asthma or asthma-related phenotypes. The aim of this study was to investigate whether a TIM-1 promoter single nucleotide polymorphism (SNP), À416 G>C, is associated with asthma in Iranian population. In this case-control study, existence of the À416 G>C polymorphism was assessed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in 300 patients with asthma (97 atopic, 203 nonatopic) and 309 healthy volunteers. Additionally, the relationship between these polymorphism genotypes and total serum IgE levels in this Iranian population was evaluated. We discovered a significant association between the À416 G>C polymorphism and atopic asthma susceptibility in the population, but this SNP showed no connection with nonatopic asthma (P < 0.05). However, our results showed significant relation between this polymorphism and serum IgE level (P < 0.05). Our results suggest that À416 G>C polymorphism in TIM-1 gene could be a predisposing factor for atopic asthma in Iranian population, and CC genotype of this SNP can be associated with increased level of IgE in patients with asthma in the same population.

TIM (T-cell immunoglobulin (Ig) and mucin domain)-1, one of the members of TIM family, expresses on Th2 cells and promotes the production of Th2 signature cytokines. This can increase a series of responses in these cells which could be one of the causes of asthma or asthma-related phenotypes. The aim of this study was to investigate whether a TIM-1 promoter single nucleotide polymorphism (SNP), À416 G>C, is associated with asthma in Iranian population. In this case-control study, existence of the À416 G>C polymorphism was assessed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in 300 patients with asthma (97 atopic, 203 nonatopic) and 309 healthy volunteers. Additionally, the relationship between these polymorphism genotypes and total serum IgE levels in this Iranian population was evaluated. We discovered a significant association between the À416 G>C polymorphism and atopic asthma susceptibility in the population, but this SNP showed no connection with nonatopic asthma (P < 0.05). However, our results showed significant relation between this polymorphism and serum IgE level (P < 0.05). Our results suggest that À416 G>C polymorphism in TIM-1 gene could be a predisposing factor for atopic asthma in Iranian population, and CC genotype of this SNP can be associated with increased level of IgE in patients with asthma in the same population.

Highlights  This study is the first of its sort in Iran which focuses on the genetic study of a very rare autosomal recessive non-syndromic nail dysplasia (NDNC) in a large kindred from Iran  The results introduce a novel pathogenic variant in the FZD6 gene in NDNC patients.  The study confirms that phenotype-directed-genotype diagnosis is an efficient approach in the diagnosis of NDNC.  The molecular findings are useful in downstream clinical utilities such as cascade genetic screening, genetic counselling and PGD.

Background and aim: SCN1A gene encodes for neuronal voltage-gated sodium-channel αsubunit. Mutations in this gene are the major cause of severe myoclonic epilepsy of infancy (Dravet syndrome) and generalized epilepsy with febrile seizures plus (GEFS +). GEFS + is a heritable benign type of epilepsy associated with febrile seizures which belongs to Idiopathic Generalized Epilepsies with a marked clinical and genetic heterogeneity. The main objective of this research is screening of mutations in scn1a gene in patients affected by GEFS + and Idiopathic Generalized Epilepsy (IGE). Methods: Genetic counseling was carried out with 30 patients and their family. Peripheral blood samples were collected from patients and DNA was extracted using salting out method. Standard PCR on 16 th-26 th exons of scn1a gene was optimized by employment of specific primers. PCR products were analyzed by SSCP in denaturant condition and sequenced in the next step. Results: Results showed a 4289c>g missense mutation in one patient affected by idiopathic generalized epilepsy. This mutation changes the alanine residue in 1430 position to glycine (A1430G). Conclusion: More studies are needed to identify the direct role of this mutation in pathogenesis, however, heterozygotic genotype of this mutation is consistent with dominant feature of inheritance of Epilepsy.

TIM (T-cell immunoglobulin (Ig) and mucin domain)-1, one of the members of TIM family, expresses on Th2 cells and promotes the production of Th2 signature cytokines. This can increase a series of responses in these cells which could be one of the causes of asthma or asthma-related phenotypes. The aim of this study was to investigate whether a TIM-1 promoter single nucleotide polymorphism (SNP), À416 G>C, is associated with asthma in Iranian population. In this case-control study, existence of the À416 G>C polymorphism was assessed using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in 300 patients with asthma (97 atopic, 203 nonatopic) and 309 healthy volunteers. Additionally, the relationship between these polymorphism genotypes and total serum IgE levels in this Iranian population was evaluated. We discovered a significant association between the À416 G>C polymorphism and atopic asthma susceptibility in the population, but this SNP showed no connection with nonatopic asthma (P < 0.05). However, our results showed significant relation between this polymorphism and serum IgE level (P < 0.05). Our results suggest that À416 G>C polymorphism in TIM-1 gene could be a predisposing factor for atopic asthma in Iranian population, and CC genotype of this SNP can be associated with increased level of IgE in patients with asthma in the same population.

Background: Autosomal recessive non-syndromic hearing loss (ARNSHL), one of the global public health concerns, is marked by a high degree of genetic heterogeneity. The role of GJB2, as the most common cause of ARNSHL, is only &lt;20% in the Iranian population. Here, we aimed to determine the relative contribution of several apparently most common loci in a cohort of ARNSHL Iranian families that were negative for the GJB2 mutations. Methods: Totally, 80 Iranian ARNSHL families with 3 or more affected individuals from Isfahan and Hamedan provinces, Iran were enrolled in 2017. After excluding mutations in the GJB2 gene via Sanger sequencing, 60 negative samples (30 families from each province) were analyzed using homozygosity mapping for 10 ARNSHL loci. Results: Fourteen families were found to be linked to five different known loci, including DFNB4 (5 families), DFNB2 (3 families), DFNB7/11 (1 family), DFNB9 (2 families) and DFNB3 (3 families). Conclusion: Despite the high heterogeneit...

MiR-21 and miR-451 are closely associated with tumor initiation, drug resistance, and recurrence of breast cancer (BC). This study was conducted to evaluate the possible value of the plasma level of miR-21 and miR-451 as potential biomarkers for the detection of primary and recurrent BC. Patients and methods: In this descriptive-analytical study, the plasma level of miR-21 and miR-451 was measured in 23 primary BC patients, 24 recurrent (local/distant metastasis) BC patients, and 24 aged-match women as healthy controls using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Finally, data were analyzed using SPSS software, and the area under the receiver operating characteristic (ROC) curve of miRNAs was measured. Results: The plasma level of miR-21 was significantly increased in both groups of primary (P<0.001) and recurrent (P<0.001) BC patients in comparison with healthy women. However, the plasma level of miR-451 was not significantly changed in primary (P=0.065) and recurrent (P=0.06) BC patients than healthy controls. The elevation of both miR-21 and miR-451 plasma level was not significantly changed in recurrent patients compared with non-recurrent (primary) patients (P=0.481, and P=1, respectively). Based on the ROC analyses, the areas under the curves (AUC) for miR-21 in discriminating primary BC and recurrent BC patients from healthy controls were 0.828 (95% CI: 0.712 to 0.944) and 0.865 (95% CI: 0.756 to 0.974), respectively. Conclusion: These data indicating that plasma miR-21 may be useful as a biomarker for the detection of both primary and recurrent BC. However, plasma miR-451 lacks enough sensitivity in the detection of primary and recurrent BC, and more studies are needed in this area.

Background and aim: The incidence of pre-lingual deafness is about 1 in 1000 neonates from which more than 60% of cases are inherited. Deafness is a heterogeneous disorder and may be due to genetic or environmental cause or both. Mutations in the DFNB59 gene ...

The need for a complementary short-term mutagenicity bioassay with robust endpoints to the Ames assay has become increasingly crucial to in order to avoid false negative results. The alternative shortterm test (STT) used in conjunction with the Ames increases the validity and decreases the number of false positive outcomes. As a result, Caco-2 cells (Human intestinal epithelial cell model) and RAW264.7 cells (mouse microphage-like cell line) were treated for 24 h with graded doses of hydrogen peroxide (0, 5, 10, 20, and 40 μM) (oxidative stress-inducing mutagen). Singleand double-strand DNA damage was quantified using single-cell gel electrophoresis (Comet assay). The head intensity, tail intensity, tail migration, and tail moment of the damaged DNA were analysed using an epifluorescence microscope with a gated camera and installed comet IV image analysis software. In Caco-2 and RAW264.7 cells, a significant drop in head intensity and a corresponding dose-dependent increase in tail ...

Cytogenetic data can be useful for taxonomic and phylogenetic studies, as well as to provide information about chromosome evolution. Therefore, it may help design conservation priorities for some threatened species, such as anurans. Herein, we describe the karyotypes of Scinax constrictus and Ololygon centralis, native endemic species from the Brazilian Cerrado. Chromosome preparations for both species were stained with Giemsa for morphological analyses and then impregnated by the Ag-NOR method for localization of the nucleolar organizer region (NOR). Both species had 24 chromosomes, as confirmed by meiotic analyses, which showed 12 bivalents. Chromosome morphologies presented the same pattern for Scinax and Ololygon compared to species already karyotyped in both genera. The NOR was interstitially located in the long arm of pair 7 in S. constrictus, whereas in O. centralis it was found near the centromere in the long arm of pair 1, thus diverging from what is commonly found for other Ololygon species. Therefore, we provide the first description of the karyotype of O. centralis and the first report of the localization of the NOR for the karyotype of both species. Our study increases the cytogenetic knowledge in species of the genera Scinax and Ololygon, and provide support for further studies on the taxonomy, ecology, and evolution of hylid anurans.

Caffeine inheritance was investigated in F 2 and BC 1 F 1 generations between Coffea arabica var. Bourbon Vermelho (BV) and Coffea canephora var. Robusta 4x (R4x). The caffeine content of seeds and leaves was determined during 2004 and 2005. Microsatellite loci-markers were used to deduce the meiotic pattern of chromosome pairing of tetraploid interspecific hybrids. Genetic analysis indicated that caffeine content in seeds was quantitatively inherited and controlled by genes with additive effects. The estimates of broad-sense heritability of caffeine content in seeds were high for both generations. In coffee leaves, the caffeine content (BSH) from the same populations showed transgressive segregants with enhanced levels and high BSH. Segregation of loci-markers in BC 1 F 1 populations showed that the ratios of the gametes genotype did not differ significantly from those expected assuming random associations and tetrasomic inheritance. The results confirm the existence of distinct mechanisms controlling the caffeine content in seeds and leaves, the gene exchange between the C. arabica BV and C. canephora R4x genomes and favorable conditions for improving caffeine content in this coffee population.

Herein, genetic relationships among five breeds of Bulgarian sheep were estimated using microsatellite markers. The total number of alleles identified was 226 at the 16 loci examined. D A distance values were used for phylogenetic tree construction with the UPGMA algorithm. The two Tsigai and two Maritza populations were found to be geneticallvery closely related to each other y (0.198, and 0.258 respectively). The Pleven Black Head population was distinct from the other four. These results could be useful for preserving genes in these breeds, thereby ensuring their preservation in Bulgaria.

Five restriction site polymorphisms in the b-globin gene cluster (HincII-5'e, HindIII-G g, HindIII-A g, HincII-yb1 and HincII-3'yb1) were analyzed in three populations (n = 114) from Reconcavo Baiano, State of Bahia, Brazil. The groups included two urban populations from the towns of Cachoeira and Maragojipe and one rural Afro-descendant population, known as the "quilombo community", from Cachoeira municipality. The number of haplotypes found in the populations ranged from 10 to 13, which indicated higher diversity than in the parental populations. The haplotypes 2 (+ ----), 3 (----+), 4 (-+ --+) and 6 (-+ + -+) on the b A chromosomes were the most common, and two haplotypes, 9 (-+ + + +) and 14 (+ + --+), were found exclusively in the Maragojipe population. The other haplotypes (1, 5, 9, 11, 12, 13, 14 and 16) had lower frequencies. Restriction site analysis and the derived haplotypes indicated homogeneity among the populations. Thirty-two individuals with hemoglobinopathies (17 sickle cell disease, 12 HbSC disease and 3 HbCC disease) were also analyzed. The haplotype frequencies of these patients differed significantly from those of the general population. In the sickle cell disease subgroup, the predominant haplotypes were BEN (Benin) and CAR (Central African Republic), with frequencies of 52.9% and 32.4%, respectively. The high frequency of the BEN haplotype agreed with the historical origin of the afro-descendant population in the state of Bahia. However, this frequency differed from that of Salvador, the state capital, where the CAR and BEN haplotypes have similar frequencies, probably as a consequence of domestic slave trade and subsequent internal migrations to other regions of Brazil.

Haptoglobin is a plasma hemoglobin-binding protein that limits iron loss during normal erythrocyte turnover and hemolysis, thereby preventing oxidative damage mediated by iron excess in the circulation. Haptoglobin polymorphism in humans, characterized by the Hp *1 and Hp *2 alleles, results in distinct phenotypes known as Hp1-1, Hp2-1 and Hp2-2, whose frequencies vary according to the ethnic origin of the population. The Hp *1 allele has two subtypes, Hp *1F and Hp *1S , that also vary in their frequencies among populations worldwide. In this work, we examined the distribution frequencies of haptoglobin subtypes in three Brazilian population groups of different ethnicities. The haptoglobin genotypes of Kayabi Amerindians (n = 56), Kalunga Afro-descendants (n = 70) and an urban population (n = 132) were determined by allele-specific PCR. The Hp *1F allele frequency was highest in Kalunga (29.3%) and lowest in Kayabi (2.6%). The Hp *1F /Hp *1S allele frequency ratios were 0.6, 1.0 and 0.26 for the Kayabi, Kalunga and urban populations, respectively. This variation was attributable largely to the Hp *1F allele. However, despite the large variation in Hp *1F frequencies, results of F ST (0.0291) indicated slight genetic differentiation among subpopulations of the general Brazilian population studied here. This is the first Brazilian report of variations in the Hp *1F and Hp *1S frequencies among non-Amerindian Brazilians.

The distribution of glutathione S-transferase (GST) GSTM1 and GSTT1 null phenotype frequencies in two Brazilian Amerindian tribes, the Munduruku tribe from Missão Cururu village (79 individuals) and the Kayabi tribe (41 individuals), was analyzed by polymerase chain reaction (PCR) amplification. The GST null phenotype frequencies for the Munduruku sample were 0% for GSTM1 and 27% for GSTT1 while for the Kayabi sample the null phenotype frequencies were 27% for GSTM1 and 29% for GSTT1. This is the first report of the absence of the GSTM1 null phenotype in any ethnic group.

Alleviation of doxorubicin (DOX)-induced cytotoxicity and genotoxicity by leaf extract of bael also known as Aegle marmelos (AME) was studied in cultured Chinese hamster V79 cells. The optimum protective dose of AME was determined by treating V79 cells with different concentrations of AME before exposure to 10 µg/ml DOX and then by evaluating the cell survival and micronuclei frequency in the cytokinesis blocked V79 cells. MTT assay results revealed that AME pretreatment resulted in a concentration dependent elevation in the cell survival up to 25 μg/ml, whereas a further increase in AME concentration reduced the cell survival. Assessment of DNA damage by micronuclei assay showed that 25 µg/ml AME reduced the micronuclei frequency to a maximum extent. Therefore, 25 μg/ml AME was considered as an optimum chemo-protective concentration and further studies were carried out using this concentration, where V79 cells were treated with 25 μg/ml AME before exposure to different concentratio...

Orofacial clefts (OFC; MIM 119530) are among the most common major birth defects. Here, we carried out mutation screening of the PVR and PVRL2 genes, which are both located at an OFC linkage region at 19q13 (OFC3) and are closely related to PVRL1, which has been associated with both syndromic and non-syndromic cleft lip and palate (nsCLP). We screened a total of 73 nsCLP patients and 105 non-cleft controls from the USA for variants in PVR and PVRL2, including all exons and encompassing all isoforms. We identified four variants in PVR and five in PVRL2. One non-synonymous PVR variant, A67T, was more frequent among nsCLP patients than among normal controls, but this difference did not achieve statistical significance.